Cells are able to process an enormous amount of infonnafion about their environment and their internal status via highly regulated signaling pathways. Critical to this information transfer are small molecule second messengers, such as cAMP, Ca2+, and a recently emerging family of phosphorylated inositides. Among the many derivatives of the inositol phosphate messengers is an intriguing subgroup that possesses high energy diphosphate groups, namely the diphosphoinositol phosphates (PP.-IPs), which have recently been connected to several cellular functions, including vesicular trafficking and apoptosis. The PP-IPs have also been linked to abnormal physiological processes including diminished insulin secretion and cancer. Owing to their chemically complex nature, there exist significant technical challenges to uncover inositol phosphate function using standard cell biology techniques. This proposal seeks to overcome these hurdles using chemical methods, to elucidate the discrete signaling functions of the PP-IPs. During the independent period of this award, aims '1,2, and 3 will rely on the deveiopment of chemical probes to study PP-IP function. Through the development of a reagent for the affinity purification of diphosphorylated proteins, the impact of this novel post-translational modification will be evaluated (aim 1). PP-IP analogues that can be covalently linked to their protein binding partners will serve to map the preferred inositol phosphate binding sites (aim 2). Lastly, aim 3 will explore the metal binding properties of the PP-IPs, which vwll then be exploited for the development of luminescent probes for in vivo imaging. Importantly, the tools from aims 1-3 will be evaluated in the cell models that have been generated during the mentored phase. The genetic interaction data will serve as a platform to generate and validate hypotheses about PP-IP signaling functions. The insulinoma cells are a critical model system to understand the role of PP-IPs in insulin secretion. By combining the chemical reagents with these genefic analyses, it will be possible to assess the physiological relevance of PP-IP signaling.